The detection of bacterial pathogens in clinical, food, dairy, environmental, and water samples frequently involves growth of the particular microorganism of interest in at least two types of culture enrichment broths. Initially, the sample is added to one type of broth and then allowed to incubate for a certain period of time at a particular temperature. A volume of this broth will then be added into a second type of enrichment broth and then incubated for a certain period of time at a particular temperature. The purpose of the enrichment process is to permit the growth of extremely low levels of a pathogen in a sample to sufficient levels for detection. For example, there may be only one viable Salmonella cell in a 25-gram food sample. Thus, there is a need to allow for growth of the Salmonella to high enough concentrations to permit detection in assays, such as pure culture assays, immunoassays, motility immobilization assays, and hybridization probe assays. The most sensitive immunoassays and hybridization assays generally require the presence of at least 100,000 cells per ml and preferably 10,000,000 cells per ml in an enrichment culture.
The U. S. Food and Drug Administration recommends microbial testing of certain food products. For example, Salmonella testing of certain food products is specified in some FDA regulations. The Division of Microbiology in the Center for Food Safety and Applied Nutrition of the U.S. Food and Drug Administration endorses a Salmonella assay procedure described in Andrews et al., "Isolation and Identification of Salmonella Species," Chapter 7 in Bacteriological Analytical Manual. 6th Edition, Association of Official Analytical Chemists, Arlington, Va. (1984). In this procedure, at least two enrichment steps are described. The first step is a "pre-enrichment step" wherein the food sample is enriched in a nutritious, nonselective broth medium to restore injured Salmonella cells to a stable condition and to promote growth. Next, a sample of the pre-enrichment broth (i.e., first enrichment broth) is added to two selective enrichment broths (i.e., second enrichment broth), wherein the sample is further enriched in a growth-promoting medium containing selective reagents. Each selective enrichment broth allows a continued increase of Salmonella organisms while simultaneously restricting proliferation of most other bacteria. The enrichment steps described in the FDA procedure both involve manual transfer of potentially biohazardous material.
The Association of Official Analytical Chemists' (AOAC) accepted method for the detection of Salmonella in a processed food sample with an immunoassay detection procedure requires that 25 grams of food sample be placed into 225 mls of a pre-enrichment broth and incubated for 24.+-.2 hours at 35.degree. C. This pre-enrichment step involves placing the sample into a nutritious medium that promotes the resuscitation and growth of the pathogen. After incubation, small aliquots of the pre-enrichment broth are transferred to two selective broths, each of which contains certain chemical agents that permit the growth of the pathogen of interest while impeding the growth of other competitor bacteria which may have grown to high concentrations in the pre-enrichment broth. In the case of Salmonella, a 1 ml portion of the pre-enrichment culture is transferred to 10 mls of tetrathionate broth and 10 mls of selenite cystine broth. The broths are incubated for 6 to 8 hours at 35.degree. C. This constitutes the selective enrichment part of the procedure. A third step, a post-enrichment step, is used for the purpose of further promoting the growth of the pathogen of interest and removing materials that may interfere with the detection assay. In the case of Salmonella, 1 ml from both the tetrathionate broth and the selenite cystine broth is transferred to separate test tubes containing sterile m-Broth. These tubes are then incubated for 14 to 18 hours at 35.degree. C. This step constitutes the post-enrichment part of the Salmonella procedure.
After the two or more enrichment steps have been completed, an immunoassay procedure, a hybridization probe assay, standard plating assays (i.e., pure culture assays), or a motility immobilization assay is conducted to detect the presence of the microorganism of interest.
The problem of multiple transfers of biohazardous material has long been recognized in the art. For example, Banwart, "Glassware Apparatus for Determining Motile Bacteria," Poultry Sci. 47:1209-1212 (1967), refers to a glass apparatus with three side U-tubes projecting into a central chamber for Salmonella assays. The side U-tubes communicate via a highly porous barrier with the central chamber containing the sample in a pre-enrichment broth. The motile Salmonella organisms were able to swim up into the side U-tubes and were able to be detected there. The benefit of the Banwart U-tube is completion of the assay, including enrichment, in about 48 hours. However, there were problems associated with the sensitivity of detection and with competing motile bacteria.
Current enrichment incubation time periods designed around the laboratory worker's standard eight-hour work day. This usually provides for full-day or overnight incubations of each step. It is likely that some of the incubation steps do not require as long an incubation period as standard microbial procedures specify. Thus, there is a need in the art for an automated enrichment device that can reduce analysis time by not requiring a laboratory technician to make transfers during nonstandard working hours.
Current immunoassay and hybridization probe assays have increased the sensitivity level for microbial detection significantly. However, there is a need for an enrichment procedure that can be automated in order to avoid transfer of potentially biohazardous material prior to conducting the immunoassay or hybridization assay detection procedure. There is also a need for an apparatus and procedure that allows for all the enrichment steps and the detections steps to be conducted in a single chamber without transfer of biohazardous material.